Solution for fast visualization of protein

ABSTRACT

The present invention provides an improved solution for staining proteins in and/or on a solid matrix or support. Typically, a protein-containing gel or membrane may be washed in a hot solution of water for about five minutes, stained in a hot solution of COOMASSIE brilliant blue dye in dilute aqueous mineral acid for about five minutes, and then rinsed in water. The washing and/or staining steps may be performed by placing the gel in a wash and/or staining solution, respectively, heating the solution in which the gel is placed to boiling in a microwave oven, and incubating the gel in the solution for about five minutes. The entire procedure can be performed in a little over ten minutes, which represents an enormous time savings over existing methods.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation of U.S. application Ser. No. 09/417,298, filedOct. 13, 1999 and now allowed.

This application claims all benefits accruing under 35 U.S.C. 119 (e)from the provisional applications U.S. application No. 60/103,903, filedOct. 13, 1998, and U.S. application No. 60/127,539, filed Apr. 2, 1999.The contents of all U.S. patents, patent applications, and otherpublications are incorporated by reference herein.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the field of detection of proteinsusing synthetic dyes and, more particularly, to the detection ofproteins in electrophoretic gels.

2. Description of the Related Art

One of the most commonly used and valuable methods for the separationand analysis of proteins is sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE). In this method, a sample containing proteinis separated by electrophoresis in a sodium dodecyl sulfate-containingbuffer through a polyacrylamide gel. As proteins are generallycolorless, the gel is usually stained after electrophoresis to visualizethe proteins.

A variety of methods are available for staining the proteins in such agel. All of these methods, however, suffer from disadvantages that areaddressed by the present invention.

For example, U.S. Pat. No. 4,555,490 (Merril) entitled “RapidVisualization System for Gel Electrophoresis” describes aphotodevelopment method involving silver ions. The procedure involvesfixing the gel with methanol/acetic acid/citric acid solution, and usinga methanolic silver nitrate solution. Methanol/acetic acid solutionshave a considerable odor. This fixing solution is apparently essentialprior to silver staining. Particular care must also generally be takento avoid contact with silver nitrate solutions. Moreover, in addition tothe contact and disposal problems associated with the use of silver ion,some care must apparently be taken to prevent silver salts fromprecipitating on the surface of the gel.

A number of methods use one of the dyes known as COOMASSIE brilliantblue to stain proteins. For example, U.S. Pat. No. 4,219,337 (Grossberget al.) entitled “Assay for Proteins and Polypeptides” describes the useof COOMASSIE brilliant blue G250 in perchloric or hydrochloric acid,which couples with the protein and undergoes a color change. This patentdoes not describe the use of this reagent to stain proteins inpolyacrylamide gels.

U.S. Pat. No. 4,946,794 (Berube) entitled “Visualization of Proteins onElectrophoresis Gels Using Planar Dyes” describes the use of COOMASSIEbrilliant blue R250 or other dyes to stain proteins in polyacrylamidegels. The procedure requires a staining step in a methanol/acetic acidsolution of the dye for one hour and a 15 minute potassium dichromatetreatment to complex the dye and differentiate between thepolyacrylamide matrix and the protein-containing spot. Thus, thisprocedure also requires the unpleasant use of methanol/acetic acid, andrequires a one hour staining step.

U.S. Pat. No. 5,273,906 (Shultz et al.) entitled “Protein StainingCompositions and Methods” describes a series of derivatives of COOMASSIEdyes which are designed to overcome some of the problems usuallyassociated with the use of COOMASSIE dyes in SDS-PAGE gels. However, instaining gels with these dyes, the staining solution is a methanolicsolution, involving the problems associated with handling and disposalof methanol. Moreover, COOMASSIE brilliant blue dyes are more widelyavailable and inexpensive than the derivatized COOMASSIE dyes of theShultz patent.

In addition to the aforementioned patents, an article by Nivinskas etal. (Bio Techniques 20:380-385, 1996) entitled “Environmentally BenignStaining Procedure for Electrophoresis Gels Using COOMASSIE BrilliantBlue” describes a procedure for staining gels with dilute aqueoussolutions of COOMASSIE brilliant blue R. The SDS-PAGE gel was typicallyfirst rinsed and washed in a large volume of dilute HCl for two hours.The gel could be boiled in water, then rinsed, but this produced nodifference in staining. The staining step involved overnight staining bya 0.0015% (w/v) solution of COOMASSIE brilliant blue G-250 in 1 mM HCl.Bands begin to appear after about one hour but the gel must be stainedovernight (i.e., 16 hours) for maximal staining and quantitation, thendestained with a large volume of 1 mM HCl and absorbent tissue wipes toabsorb unbound dye. Although this method eliminates the problemsassociated with methanol and acetic acid, it is still slow and requiresseveral solution changes.

Thus, I have noted that the existing methods of staining gels suffereither from problems of use of undesirable reagents, such as acetic acidand methanol or silver nitrate, or from long processing times.Additionally, I have noticed that some of the methods are inconsistentin the staining results, which can lead to the ruining of an experiment.Based on my reading of the contemporary art, I have determined that whatis needed is a faster and easier way of staining proteins in and/or onsolid matrices and supports.

SUMMARY OF THE INVENTION

It is therefore an object of my invention to provide an improved methodof staining proteins embedded in and/or on a solid matrix (e.g., a gelor porous particle) or solid support (e.g., a membrane or porousfilter).

It is also an object of the invention to provide a method of stainingproteins which takes only a very short time (i.e., less than about onehour, 30 minutes, or 15 minutes).

It is a further object of the invention to provide a method of stainingproteins which uses aqueous solutions.

It is a yet further object of the invention to avoid the use of certainnoxious alcohols (e.g., methanol) and/or organic acids (e.g., aceticacid) as the solvent of the solutions being utilized, or a majorcomponent thereof.

It is a still further object of the invention to provide a stainingmethod which consistently and reproducibly stains proteins.

It is a still further object of the invention to avoid the use of aspecial fixing step (e.g., acetic acid as a fixative) before proteinstaining.

These objects as well as other which will be apparent from thedescription below are achieved by the invention which provides a methodfor staining immobilized proteins (e.g., immobilized in a polyacrylamideslab gel, in an agarose or polyacrylamide bead, on and/or in a flexiblemembrane, on and/or in a disc or fiber filter) using at least one or twobrilliant blue stains. This method may include the steps of: washing theimmobilized protein(s) on and/or in a solid matrix or support in hotwash solution, staining the immobilized protein(s) in a hot stainingsolution containing brilliant blue dye in dilute acid, and rinsing thesolid matrix or support to rinse the dye which is not associated withthe immobilized protein(s). Washing and destaining steps may beconsidered as optional for performing the method of the invention, butthey may improve the results obtained.

The wash solution may be water or a dilute acid (e.g., hydrochloric orperchloric acid). By “hot” is meant a solution heated to substantiallyabove room temperature and the solution may cool thereafter or bemaintained at the initial temperature. The hot wash solution mayinitially be at a temperature greater than 45° C., greater than 90° C.,or brought to a boil. The washing step may be achieved by placing thegel in the wash solution and then removing the solution at the end ofthe incubation period. The wash solution may be heated in a microwaveoven or on a hot plate, before or after the gel is placed therein, untilthe desired temperature is achieved.

Likewise, the staining solution is heated to substantially above roomtemperature; the hot staining solution may initially be at a temperaturegreater than about 45° C., greater than about 70° C., greater than about90° C., or brought to a boil. The staining step may be achieved byplacing the gel in the staining solution and then removing the solutionat the end of the incubation period. The staining solution may be heatedin a microwave oven or on a hot plate, before or after the gel is placedtherein, until the desired temperature is achieved.

For a solid support (e.g., a membrane or filter), the entire proceduremay be done at room temperature. Here, washing might not be required.But, as in the applications involving a solid matrix, extended rinsing(e.g., long incubation time, agitation, multiple changes of rinsingsolution) may be used to destain the background staining initiallypresent in and/or on the support or matrix after staining and therebyachieve increased sensitivity or reduction in the background. Thus,destaining may be useful but might not be required.

The brilliant blue dye may be used at a concentration in the range ofabout 0.0005 to 0.5% (w/v), preferably about 0.001 to 0.05% (w/v), indilute acid. The dilute acid may be hydrochloric or perchloric acid; theconcentration of dilute acid may be less than the equivalent of about100 mM HCl, preferably in the range between about 5 and 50 mM HCl, andmore preferably in the range between about 10 and 35 mM HCl. Thestaining solution may contain COOMASSIE brilliant blue G-250 at a finalconcentration of about 0.006% (w/v) in about 35 mM HCl or COOMASSIEbrilliant blue R-250 at a final concentration of about 0.0005% (w/v) inabout 10 mM HCl.

The solid matrix or support which has been stained in accordance withthis method is also an object of the invention. In particular, such aproduct will not have been exposed to methanol and/or acetic acid duringthe process. The protein(s) immobilized on and/or in the product can bequickly processed for further analysis or storage. Preferred is aproduct in which staining of immobilized protein(s) is maximal and/orquantitative.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is a staining procedure for proteins, especiallythose in and/or on gels, porous particles, membranes, and filters. Thereagent for performing the procedure has been sold under the tradenameSpeedStain as a kit for staining protein gels.

The present invention makes use of the dye compounds known as brilliantblue R and brilliant blue G, which are well known in the art and areavailable from a number of sources. They are also known by thetradenames COOMASSIE brilliant blue R and COOMASSIE brilliant blue G.These dyes are generally available as monosodium salts, although thenature of the counterion does not appear to be important. Brilliant blueG has Chemical Abstracts registry number [6104-58-1] and brilliant blueR has Chemical Abstracts registry number [6104 -59-2]. Here, moresuccess has been achieved with brilliant blue dye obtained from Bio-RadLaboratories (Hercules, Calif.).

In the present patent application, the dyes brilliant blue R andbrilliant blue G will be referred to as brilliant blue dyes. These dyesare available as powder or dissolved in ethanolic solutions and thesesolutions are often used to prepare methanolic staining solutions.Brilliant blue R has been used in strongly acidic solutions, such as12.5% trichloroacetic acid, in which the dye is generally colloidal.Brilliant blue R is apparently even less soluble in acid than isbrilliant blue G. In the present application, solutions of the brilliantblue dyes in dilute aqueous acids will be referred to as colloidalbrilliant blue solutions. The colloidal and non-colloidal forms might beseparated by filtration. Most or as much as 95% of the brilliant bluedye may be in the colloidal form when the staining solutions areprepared according to the present invention.

The present invention uses staining solutions of the brilliant blue dyesin a dilute acid solution with water or an aqueous buffer as thesolvent. Preferably, the brilliant blue dye is dissolved in a dilutemineral acid solution. The acid used may be hydrochloric acid;alternatively, the acid used may be perchloric acid. It has been found,however, that phosphoric acid is not preferred. Alternatively,trichloroacetic acid (TCA) might also be used.

A typical brilliant blue G staining solution used in the presentinvention was made as follows: dissolve COOMASSIE brilliant blue G-250in deioinized water to achieve a final concentration of about 0.006%(w/v), stir the solution for about two hours at room temperature, andadd concentrated hydrochloric acid to achieve a final concentration ofabout 35 mM HCl. Preferably, the dilute acid solution is equivalent tomore than about 10 mM HCl and/or less than about 50 mM HCl.

A typical brilliant blue R staining solution used in the presentinvention was made as follows: dissolve COOMASSIE brilliant blue R-250in deioinized water to achieve a final concentration of about 0.0005%(w/v), stir the solution for about 24 hours at room temperature, and addconcentrated hydrochloric acid to achieve a final concentration of about10 mM HCl. Preferably, the dilute acid solution is equivalent to morethan about 5 mM HCl and/or less than about 50 mM HCl.

The staining solutions used in the present invention may have aconcentration of brilliant blue dye of between about 0.0005 and 0.5%(w/v); preferably, the concentration is between about 0.001 and 0.05%(w/v). A typical concentration of brilliant blue G is about 0.006% (w/v)and a typical concentration of brilliant blue R is about 0.0005% (w/v),but other concentrations may be used as well.

Hydrochloric acid or perchloric acid may be used as the dilute aqueousacid, and the dilute acid is preferably at a concentration in the rangebetween about 1 and 50 mM. A preferred concentration of acid is betweenabout 10 and 35 mM.

In the present application, the term “protein gel” refers to a gel inwhich proteins have been electrophoresed. The invention can also be usedfor gels in which the proteins have been separated by a method known asisoelectric focussing and for 2-D gels. The matrix may also be a porousparticle. Additionally, the invention can be used to stain proteins onand/or in membranes or filters made from natural or artificial materialssuch as cellulose or derivatized versions thereof (e.g., nitrocellulose)and nylon or derivatized versions thereof (e.g., PVDF). The protein mayalso be applied to the solid matrix or support by capillary action orwicking, chromatography, electrophoresis or electrofocussing, or othermethods such as, for example, western blotting and immunoblotting.

After electrophoresis is completed, the minigel was immersed in about200 ml of deionized or distilled water or dilute acid, for example in aglass or plastic tray. The gel and the wash solution were placed in amicrowave oven, heated until the solution boiled, and then washed forabout five minutes. Alternatively, the tray containing the gel and washsolution may be placed on a hot plate and heated until the solutionboils; heating may be stopped once the solution boils and the gel isallowed to incubate for about 5 minutes. Washing may remove materialswhich interfere with protein staining (e.g., SDS). The wash solution andthe gel may be separated to remove the solution. The gel may betransferred from the wash solution to accomplish this, but generally thesolution will be poured or aspirated away from the gel. Then, about 20to 50 ml of a staining solution having a brilliant blue dye in diluteacid solution, was added to the gel.

The gel and the staining solution were placed in a microwave oven andheated until the solution boils. Typically, this took about 30 to 60seconds but this may vary depending on the volume of solution and thepower of the microwave oven. Alternatively, the tray containing the geland brilliant blue solution may be placed on a hot plate and heateduntil the solution boils; heating may be stopped once the solutionboils. The gel was incubated in the brilliant blue solution for aboutfive minutes. The staining solution is preferably used only once becauseI have found that reuse results in a dramatic loss of sensitivity.

The brilliant blue solution was then removed from around the gel, andthe gel was placed in deionized or distilled water for rinsing. Theprotein bands on the gel were now stained and visible to the eye asintense light blue bands (i.e., sky blue in the present invention incontrast to dark blue for conventional COOMASSIE staining). The gel isready for photography, examination by eye, measurement of lighttransmission or adsorbance by an optical instrument, or othervisualization methods. The gel may also be used for other furtherstudies, such as drying or transfer to a solid support. If there is asufficient amount of protein in the band, the rinsing solution merelywashes the staining solution off the surface of the gel because nodestaining (i.e., removal of brilliant blue dye trapped within the gel)is needed. But a light background in the gel may develop, and where afaint protein band is to be detected, this background may be removed byovernight washing.

Typically, in the present invention, protein bands containing as littleas 20 ng are visible as intense blue bands against a light bluebackground. Optionally, after staining, the gel may be immersed in asolution of about 10 to 100 mM HCl acid which tends to fix the stainedbands and remove the background of brilliant blue dye in the gel. Thisbackground is a very light blue throughout the gel due to the presenceof some non-colloidal brilliant blue dye; this background can be reducedsignificantly by overnight washing. Isopropanol may intensify the colorof the stained band while methanol and ethanol do not have this effect.

A solid matrix which has been stained may then have protein bands orspots immobilized in and/or on the matrix quantitated by an opticalinstrument using light transmission or adsorbance. The protein bands orspots may also be transferred to a solid support, stained, andquantitated by an optical instrument using light transmission oradsorbance. A laser densitometer can be used to scan the solid matrix orsupport and quantitate the amount of protein by comparison to a set ofstandard amounts. Staining is considered “maximal” when the amount ofstain detected in a protein band or spot does not increase in asignificant manner (i.e., staining is saturated). Because binding ofbrilliant blue dye by protein is stoichiometric, staining is considered“quantitative” when the amount of stain detected is directly related tothe amount of protein present. Preferably, under the identical orsimilar conditions for performing the present invention, the set ofstandard amounts used to calibrate this quantitation can be plotted in alinear relationship to the amount of brilliant blue dye bound by proteinin that region where the previously unknown amount of protein needs tobe determined.

Alternatively, the washing and staining solutions may be used hot butbelow their boiling points. By “hot” is meant that the solution isheated to substantially above room temperature. For example, the hotwash solution may be at a temperature greater than about 45° C., greaterthan about 90° C., or brought to a boil. The washing step may beachieved by placing the gel in the wash solution and heating in amicrowave oven or on a hot plate until the desired temperature isachieved. Alternatively, the solution may be preheated and poured overthe gel or the gel may be transferred into the preheated solution.

Likewise, the hot staining solution is a solution heated tosubstantially above room temperature. For example, the hot stainingsolution may be at a temperature greater than about 45° C., greater thanabout 70° C., greater than about 90° C., or brought to boiling. Thestaining step may be achieved by placing the gel in the wash solutionand heating in a microwave oven or on a hot plate until the desiredtemperature is achieved. Alternatively, the solution may be preheatedand poured over the gel or the gel may be transferred into the preheatedsolution.

For staining proteins on a solid support, the entire staining proceduremay be performed at room temperature. Hot solutions are not requiredwhen there is no solid matrix which needs to be penetrated.

The technique of PAGE is well known in the art. When electrophoresis ofthe polyacrylamide gel is completed, the gel is generally removed fromthe electrophoresis apparatus for staining. PAGE may be performed withgels of various sizes, polyacrylamide concentrations, and ratios ofbis-acrylamide to acrylamide; with different electrophoretic buffersystems; in the presence or absence of sodium dodecyl sulfate (SDS). Thepresent invention is here described for a protein minigel ofpolyacrylamide at a typical concentration between about 4 and 25% usedfor protein gels. Such SDS-PAGE gels has been maximally stained in aboutfive minutes when the gel was about 1.0 mm thick, but increasing thethickness of the gel to about 1.5 mm results in a doubling of time tocomplete staining. The following example illustrates the invention butdoes not limit the legal protection of an issued patent unless aparticular limitation is explicitly recited in the claims. For example,non-denaturing or non-polyacrylamide gels could be used in practicingthe present invention under reducing and/or non-reducing conditions.

Bovine serum albumin (2 mg BSA per ml standard, Pierce) was diluted withphosphate buffered saline (PBS) to final concentrations of 1.0, 0.4,0.2, 0.1, 0.04, 0.02, 0.01, 0.004 and 0.002 mg/ml. To 30 μl of thesedilutions, 30 μl of a 2× SDS Tris-glycine sample Buffer (#LC2676, Novex)containing 10% (v/v) β-mercaptoethanol were added and incubated forthree minutes in a boiling water bath. Ten μl of each sample were thenloaded on a pre-cast 4-20% gradient polyacrylamide denaturing minigel(8×8 cm×1.0 mm; #EC60252, Novex) with 12-wells (4×9×1 mm in accordancewith the comb that was used), and electrophoresed in an XCell IIapparatus (#EI9001, Novex) using a 1:10 dilution of 10× Tris-glycine SDSrunning buffer (#LC2675, Novex) in deionized (d.i.) water at a constantvoltage of 185 V for 60 minutes. Gels were removed from the apparatusand placed in 200 ml of d.i. water in a plastic tray (Nalgene).

The gel in water was placed in a microwave oven, heated for 3 minutes,and then incubated on a rocker for five minutes. The water was pouredoff from the gel. Then 40 ml of a staining solution having brilliantblue G-250 dye in 30 mM HCl, was added to the gel.

The gel and the staining solution were placed in a microwave oven andheated for one minute. The solution started boiling. The gel wasincubated in the hot brilliant blue solution for five minutes withrocking. The brilliant blue solution was then removed from around thegel, and the gel was placed in d.i. water. The protein bands on the gelwere now stained and visible to the eye and dilutions down to 20 ng ofBSA were visible. This is similar to the sensitivity that can beachieved by conventional staining with a COOMASSIE dye.

The concentration of brilliant dye in the staining solution and/orincubation times may be increased by those skilled in the art if theamount or concentration of immobilized protein is decreased, a dimensionof the solid matrix (e.g., thickness of a gel or diameter of a bead) isincreased such that penetration by solution is decreased, or the like.

One advantage of the present invention is that the washing stepperformed before the staining step does not require acetic acid ormethanol. One wash solution which may be used may be considered toconsist essentially of water. Here, consist or consisting “essentiallyof” means that the solution is primarily water or simple aqueoussolutions, without additives like acetic acid and alcohols which willincrease the solubility of non-colloidal brilliant blue dye andtherefore increase the background. This wash solution may be distilledor deionized water, but may also be tap water which has impurities whichdo not affect the washing. Alternatively, the wash solution may be adilute perchloric or hydrochloric acid solution. Typically, a volume ofwash solution greater than about ten times the volume of the gel is usedonce and then removed prior to staining the gel.

As described above, the entire procedure can be performed in as littleas about ten minutes. The staining step of the procedure can becompleted in as little as about five minutes, at which time bands willbe visible and staining is maximal, for a 4-20% gradient polyacrylamidedenaturing gel which is about 1.0 mm thick. Under these conditions andwith destaining overnight, the sensitivity of the present invention isat least about 50 ng bovine serum albumin per mm³ gel. In contrast,increasing the thickness of the gel to about 1.5 mm, doubles the timerequired for maximal staining but does not appreciably reducesensitivity.

Therefore, the complete procedure takes considerably less time than thestandard procedures, which usually involve a minimum staining time ofabout 20 minutes, and typically take more than an hour to as long asovernight to complete. By virtue of the short and simple procedure,there is no need to monitor the gel for the level of staining over acourse of hours, which can be a great inconvenience.

Moreover, in the present procedure, there is no need to deal with thesmell or disposal problems of methanol and acetic acid solutions. Thedilute acids of the present invention may be neutralized to harmlesssolutions, and the dye may be adsorbed by charcoal, generally yielding aharmless aqueous solution for disposal. Furthermore, there is noappreciable shrinkage of the gel (i.e., much less than about 5%) usingthe present invention whereas there is on the order of 5-40% shrinkageof gels with acetic acid and methanol.

It is to be understood that the embodiments described herein areillustrative of the present invention, and that various modifications orchanges in light thereof will be suggested to those of skill in the art,and are to be included within the scope of the appended claims. In thisrespect, the patent protection provided would be determined from theallowed claims and legal equivalents thereof instead of being limited bythe specification.

I claim:
 1. A solution comprising at least one brilliant blue dye ascolloids in a dilute acid solution; wherein said dilute acid has aconcentration equivalent to between about 1 mM and 100 mM, and said atleast one brilliant blue dye has a concentration between 0.0005% (w/v)and 0.5% (w/v).
 2. The solution of claim 1, wherein said concentrationof dilute acid is less than 50 mM.
 3. The solution of claim 1, whereinsaid concentration of dilute acid is less than 35 mM.
 4. The solution ofclaim 1, wherein said concentration of dilute acid is more than 5 mM. 5.The solution of claim 1, wherein said concentration of dilute acid ismore than 10 mM.
 6. The solution of claim 1, wherein said dilute acidhas a concentration in the staining solution equivalent to between about1 and 50 mM HCl.
 7. The solution of claim 1, wherein said dilute acidhas a concentration in the staining solution equivalent to between about10 and 35 mM HCl.
 8. The solution of claim 1, wherein said concentrationof at least one brilliant blue dye is between about 0.001% (w/v) and0.05% (w/v).
 9. The solution of claim 1, wherein said at least onebrilliant blue dye is brilliant blue G-250.
 10. The solution of claim 1,wherein said at least one brilliant blue dye is brilliant blue R-250.11. The solution of claim 1, wherein said dilute acid is hydrochloricacid.
 12. The solution of claim 1, wherein said dilute acid isperchloric acid.
 13. The solution of claim 1, wherein the solution has atemperature of at least 45° C.
 14. The solution of claim 1, wherein thesolution has a temperature of at least 70° C.
 15. The solution of claim1, wherein the solution has a temperature of at least 90° C.
 16. Asolution comprising at least one brilliant blue dye as colloids in adilute mineral acid solution; wherein said dilute mineral acid has aconcentration equivalent to between about 5 mM and 100 mM, and said atleast one brilliant blue dye has a concentration between 0.0005% (w/v)and 0.5% (w/v).
 17. A method of staining protein comprising: (a)providing protein immobilized in and/or on a solid matrix, and (b)staining said protein with the solution of claim
 16. 18. The method ofclaim 17 further comprising destaining said protein with a washsolution.
 19. A membrane, wherein protein immobilized on said membraneis stained, prepared according to claim
 17. 20. A gel, wherein proteinimmobilized in said gel is stained, prepared according to claim 17.